This grant application is for the continuation of genetic studies of silencing in Saccharomyces cerevisiae, a project entering its 17th year. Accomplishments in the most recent renewal period include 1) establishment that ORC is the replication initiator in yeast, 2) discovery that the silencing and replication functions of ORC are separable, 3) establishing that replication initiation from the silencer is not required for silencing, 4) discovery that ORC bound to silencers recruits Sir1p to the silencer through binding to a specific set of amino acids in Sir1p, 5) discovery that at least some functions of ORC are conserved between yeast and Drosophila, 6) discovery of a presumptive acetyltransferase involved in silencer function, possibly through affects on ORC or origins, 7) discovery of five new proteins involved in silencing, two of which have human homologs with direct connection to leukemia. 8) discovery that Drosophila Sir2 protein is a regulator of position effects. 9) discovery of a potential M phase role for ORC 10) discovery of the first chromatin boundary element in yeast. 11) discovery of silencing proteins in K. lactis that challenge existing models for telomere length regulation, and several more discoveries. Two technical breakthroughs include the ability to visualize ORC on meiotic and mitotic chromosomes, and antibody localization of Drosophila Sir2p. In the coming period, the project will 1) determine if replication through HMR is required to establish silencing, 2) test whether orc mutants' M-phase arrest reflects a new check point or a role for ORC in promoting sister chromatid segregation, 3) use the ability to visualize ORC on metaphase chromosomes to answer fundamental questions about ORC vis-a-vis replication and silencing, 4) determine the role of newly discovered ORC binding proteins, 5) identify new conditional ORC mutations for ordering events in the cell cycle, 6) figure out how SUM1-1 mediates silencing, 7) learn how chromatin boundary elements are assembled and function, 8) test for other silenced regions in the genome, 9) determine how a presumptive protein methyltransferase affects silencing, and 10) resolve the basis of the different silencing properties of artificial and natural telomeres.